臺灣博碩士論文加值系統

流行感冒為一常見的新興病毒感染疾病，由於在該疾病的預防與治療上仍沒有合適的疫苗與藥物，因此A型流感病毒每年仍造成人們的威脅與經濟上的損失．螺旋藻為一種微小且呈絲狀的光合菌，從阿茲特克時期即被記載具有悠久的食用歷史．在過往的研究中，螺旋藻萃取物即被發現具有降低膽固醇、調節免疫、抗氧化、抗癌與抑制病毒的功效．在本研究中，我們發現螺旋藻冷水萃取物具抑制病毒的特性，且對細胞的毒性低，於動物毒理試驗中，一次給予每公斤體重5000毫克，或連續14天給予每公斤體重3000毫克的螺旋藻冷水萃取物後，大鼠對螺旋藻冷水萃取物仍具有良好的耐受性． 螺旋藻冷水萃取物被發現能抑制多種且包含具克流感抗藥性的流感病毒斑形成，其一作用機制被發現能藉由抑制流感病毒的血球凝集作用以在病毒感染細胞的早期降低病毒的複製，並能改善受流感病毒感染後的小鼠存活率．除流感病毒外，我們亦發現螺旋藻冷水萃取物於細胞實驗中能抑制東南亞地區主要的新興傳染病毒，腸病毒71型，克沙奇A16、A10與呼吸道融合病毒．從上述結果推測，螺旋藻冷水萃取物未來可能可開發為一種安全且有效的藥劑來管控流感病毒與腸病毒71型的爆發，且需進一步的以臨床試驗來驗證其臨床功效．

Influenza is the most common emerging viral diseases in the world. Since lack of optimal vaccine and drugs, influenza A virus is still a threat to human. Spirulina spp. is a microscopic and filamentous cyanobacterium, and had been used as foods from Aztec civilization. The extracts of Spirulina were reported to have several medicinal effects on cholesterol reduction, immune-modulation, antioxidant, anticancer, and anti-virus effects. In this study, we show that Spirulina water extracts have low cellular toxicity, and the cold-water extract was further demonstrated that one dose as high as 5,000 mg/kg, or 3,000 mg/kg/day for 14 successive days to animal models. The cold water extract of Spirulina had been found to inhibit viral plaque formation in a broad range of influenza viruses, including oseltamivir-resistant strains through act at an early stage of infection to reduce virus yields in cells and improve survival in influenza-infected mice, with inhibition of influenza hemagglutination identified as one of the mechanisms involved. Otherwise, we also found the Spirulina extract could inhibit enterovirus 71 (EV71), coxsackie A16, A10 viruses and respiratory syncytial virus. In conclusion, these results suggest that the Spirulina extract might serve as a safe and effective therapeutic agent to manage influenza and further clinical investigation may be warranted.

指導教授推薦書
口試委員審定書
誌 謝 iii
中 文 摘 要 iv
ABSTRACT v
Table of Contents vi
List of Figures ix
List of Tables x
CHAPTER I INTRODUCTION 1
1.1 Emerging viral diseases 1
1.2 Epidemiology of Influenza viruses 1
1.3 Spirulina (Arthrospira platensis) 3
1.4 The referenced anti-viral property of Spirulina extracts 3
1.5 Objective of the study 4
CHAPTER II MATERIALS AND METHODS 5
2.1 Cold water extraction of Spirulina (Arthrospira platensis) 5
2.2 Cell cultures and influenza viral strains 5
2.3 Plaque reduction assay 6
2.4 Cytotoxicity assay of Spirulina extract. 6
2.5 Viruses neutralization test 7
2.6 Virus yields percentage with TCID50 evaluation 8
2.7 Time of addition assay 8
2.8 Hemagglutination inhibition assay 9
2.9 Spirulina extract toxicity assays. 9
2.10 Survival analysis in a mouse model 10
2.11 Statistical analysis 11
CHAPTER III RESULTS 12
3.1 Spirulina extract inhibits plaque formation in a broad range of influenza strains. 12
3.2 Spirulina extract is safe and well-tolerated at high doses in animal model. 14
3.3 Spirulina extract disrupts viral replication most effectively during early infection. 16
3.4 Spirulina extract targets hemagglutinin to inhibit influenza virus. 17
3.5 Spirulina extract improves survival in influenza-infected mice. 19
3.6 The Spirulina extracts is able to neutralize the emerging virus, enterovirus 71, coxsackie A16 and A10 and respiratory syncytial virus A2 (RSV) 19
CHAPTER IV DISCUSSION 21
Reference 28
Appendix 33
 
List of Figures
FIGURE 1. THE COLD WATER EXTRACT OF SPIRULINA (ARTHROSPIRA PLATENSIS) INHIBITS VIRAL REPLICATION AND PLAQUE FORMATION IN A BROAD RANGE OF INFLUENZA STRAINS IN VITRO. 34
FIGURE 2. SHORT-TERM ACUTE AND SUBACUTE ORAL TOXICITY FOR THE SPIRULINA EXTRACT. 36
FIGURE 3. SPIRULINA EXTRACT ACTS TO DISRUPT VIRAL REPLICATION AT AN EARLY STAGE OF INFECTION. 37
FIGURE 4. SPIRULINA EXTRACT INHIBITS INFLUENZA VIRUS INFECTION BY DISRUPTING HEMAGGLUTINATION. 39
FIGURE 5. SPIRULINA EXTRACT IMPROVES SURVIVAL RATES IN INFLUENZA-INFECTED MICE. 41

 
List of Tables
TABLE 1. ANTIVIRAL ACTIVITY OF SPIRULINA EXTRACT AGAINST INFLUENZA VIRUS STRAINS IN VITRO. 42
TABLE 2. HEMATOLOGY RESULTS IN RATS (MEAN ± SD, N=10) 43
TABLE 3. SERUM CHEMISTRY IN RATS (MEAN ± SD, N=10) 44
TABLE 4. MUTAGENICITY TEST OF SPIRULINA EXTRACT IN SALMONELLA TYPHIMURIUM STRAINS WITHOUT S9 METABOLIC ACTIVATION 45
TABLE 5. MUTAGENICITY TEST OF SPIRULINA EXTRACT IN SALMONELLA TYPHIMURIUM STRAINS WITHOUT S9 METABOLIC ACTIVATION 46
TABLE 6. EFFECTS OF SPIRULINA EXTRACT ON THE INDUCTION OF CHROMOSOME ABERRANTIONS IN CHINESE HAMSTER OVARY CELLS FOR 3-HOUR TREATMENT IN THE ABSENCE OF S9 47
TABLE 7. EFFECTS OF SPIRULINA EXTRACT ON THE INDUCTION OF CHROMOSOME ABERRANTIONS IN CHINESE HAMSTER OVARY CELLS FOR 3-HOUR TREATMENT IN THE PRESENCE OF S9 48
TABLE 8. ANTIVIRAL ACTIVITIES OF SPIRULINA EXTRACT AGAINST VARIOUS EMERGING VIRUSES 49
TABLE 9. COMPOSITION ANALYSIS OF SPIRULINA EXTRACT 50
TABLE10. ANTIVIRAL ACTIVITY OF THE FURTHER FRACTIONATION OF SPIRULINA EXTRACT AGAINST FLU A/WSN/33 VIRUS 51

 

1. Taubenberger, J. K. & Morens, D. M. The pathology of influenza virus infections. Annu. Rev. Pathol. 3, 499–522 (2008).
2. Tamerius, J. et al. Global influenza seasonality: reconciling patterns across temperate and tropical regions. Environ. Health Perspect. 119, 439–445 (2011).
3. Johnson, N. P. & Mueller, J. Updating the accounts: global mortality of the 1918-1920 "Spanish" influenza pandemic. Bull. Hist. Med. 76, 105–115 (2002).
4. Dawood, F. S. et al. Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation: a modelling study. Lancet Infect. Dis. 12, 687–695 (2012).
5. Tong, S. et al. New world bats harbor diverse influenza A viruses. PLoS pathogens 9, e1003657, doi:10.1371/journal.ppat.1003657 (2013).
6. Neumann, G., Noda, T., & Kawaoka, Y. Emergence and pandemic potential of swine-origin H1N1 influenza virus. Nature 459, 931–939 (2009).
7. Zheng, B. J. et al. Delayed antiviral plus immunomodulator treatment still reduces mortality in mice infected by high inoculum of influenza A/H5N1 virus. Proc. Natl. Acad. Sci. USA 105, 8091–8096 (2008).
8. Geiler, J. et al. N-acetyl-L-cysteine (NAC) inhibits virus replication and expression of pro-inflammatory molecules in A549 cells infected with highly pathogenic H5N1 influenza A virus. Biochem. Pharmacol. 79, 413–420 (2010).
9. Deng, R. & Chow, T. J. Hypolipidemic, antioxidant, and antiinflammatory activities of microalgae Spirulina. Cardiovasc. Ther. 28, e33–e45 (2010).
10. Marles, R. J. et al. United States pharmacopeia safety evaluation of spirulina. Crit. Rev. Food Sci. Nutr. 51, 593–604 (2011).
11. Karkos, P. D., Leong, S. C., Karkos, C. D., Sivaji, N., & Assimakopoulos, D. A. Spirulina in clinical practice: evidence-based human applications. Evid. Based Complement. Alternat. Med. 2011, 531053 (2011).
12. Hayashi K HT, Morita N, Kojima I. An extract from Spirulina platensis is a selective inhibitor of herpes simplex virus type 1 penetration into HeLa cells. Phytotherapy Research 1993; 7: 76-80
13. Hayashi T, Hayashi K, Maeda M, Kojima I. Calcium spirulan, an inhibitor of enveloped virus replication, from a blue-green alga Spirulina platensis. Journal of natural products 1996; 59(1): 83-7.
14. Hayashi K, Hayashi T, Kojima I. A natural sulfated polysaccharide, calcium spirulan, isolated from Spirulina platensis: in vitro and ex vivo evaluation of anti-herpes simplex virus and anti-human immunodeficiency virus activities. AIDS research and human retroviruses 1996; 12(15): 1463-71.
15. Ayehunie S BA, Baba TW, Ruprecht RM. Inhibition of HIV-1 replication by an aqueous extract of Spirulina platensis (Arthrospira platensis). J Acquir Immune Defic Syndr Hum Retrovirol 1998; 18: 7-12.
16. Lee JB, Srisomporn P, Hayashi K, Tanaka T, Sankawa U, Hayashi T. Effects of structural modification of calcium spirulan, a sulfated polysaccharide from Spirulina platensis, on antiviral activity. Chemical & pharmaceutical bulletin 2001; 49(1): 108-10.
17. Shih, S. R. et al. Pyrazole compound BPR1P0034 with potent and selective anti-influenza virus activity. J. Biomed. Sci. 17, 13 (2010).
18. Fineberg, H. V. Pandemic preparedness and response — lessons from the H1N1 influenza of 2009. N Engl J Med 370, 1335–1342 (2014).
19. Ison, M. G. Antivirals and resistance: influenza virus. Curr. Opin. Virol. 1, 563–573 (2011).
20. Jefferson, T. et al. Oseltamivir for influenza in adults and children: systematic review of clinical study reports and summary of regulatory comments. BMJ. 348, g2545 (2014).
21. Simpore, J. et al. Nutrition rehabilitation of HIV-infected and HIV-negative undernourished children utilizing spirulina. Ann Nutr Metab. 49, 373–380 (2005).
22. Hirahashi, T et al. Activation of the human innate immune system by Spirulina: augmentation of interferon production and NK cytotoxicity by oral administration of hot water extract of Spirulina platensis. Int. Immunopharmacol. 2, 423–434 (2002).
23. Mao, T. K., Van de Water, J., & Gershwin, M. E. Effects of a Spirulina-based dietary supplement on cytokine production from allergic rhinitis patients. J. Med. Food 8, 27–30 (2005).
24. Cingi, C., Conk-Dalay, M., Cakli, H., & Bal, C. The effects of spirulina on allergic rhinitis. Eur. Arch. Otorhinolaryngol. 265, 1219–1223 (2008).
25. Pugh, N. D. et al. Oral administration of a Spirulina extract enriched for Braun-type lipoproteins protects mice against influenza A (H1N1) virus infection. Phytomedicine 22, 271–276 (2015).
26. Shih, S. R., Tsai, K. N., Li, Y. S., Chueh, C. C., & Chan, E. C. Inhibition of enterovirus 71-induced apoptosis by allophycocyanin isolated from a blue-green alga Spirulina platensis. J. Med. Virol. 70, 119–125 (2003).
27. Cherng, S. C., Cheng, S. N., Tarn, A., & Chou, T. C. Anti-inflammatory activity of c-phycocyanin in lipopolysaccharide-stimulated RAW 264.7 macrophages. Life Sci. 81, 1431–1435 (2007).
28. Leung, P. O., Lee, H. H., Kung, Y. C., Tsai, M. F., & Chou, T. C. Therapeutic effect of C-phycocyanin extracted from blue green algae in a rat model of acute lung injury induced by lipopolysaccharide. Evid. Based Complement. Alternat. Med. 2013, 916590 (2013).
29. Shih, C. M., Cheng, S. N., Wong, C. S., Kuo, Y. L., Chou, T. C. Antiinflammatory and antihyperalgesic activity of C-phycocyanin. Anesth. Analg. 108, 1303–1310 (2009).
30. Reddy, C. M. et al. Selective inhibition of cyclooxygenase-2 by C-phycocyanin, a biliprotein from Spirulina platensis. Biochem. Biophys. Res. Commun. 277, 599–603 (2000).
31. Hwang, J. H., Chen, J. C., & Chan, Y. C. Effects of C-phycocyanin and Spirulina on salicylate-induced tinnitus, expression of NMDA receptor and inflammatory genes. PLoS One 8, e58215 (2013).
32. Lee, S. M. et al. Hyperinduction of cyclooxygenase-2-mediated proinflammatory cascade: a mechanism for the pathogenesis of avian influenza H5N1 infection. J. Infect. Dis. 198, 525–535 (2008).